Decrease in cytosolic calcium/phospholipid-dependent protein kinase activity following phorbol ester treatment of EL4 thymoma cells.
Decrease in cytosolic calcium/phospholipid-dependent protein kinase activity following phorbol ester treatment of EL4 thymoma cells.
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佛波酯处理 EL4 胸腺瘤细胞后胞质钙/磷脂依赖性蛋白激酶活性降低。
DOI：
10.1016/s0021-9258(18)33427-6
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
J. J. Sando
中科院分区：
文献类型：
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作者：
A. Kraft;W. Anderson;H. Cooper;J. J. Sando
方法描述了EL4细胞的细胞来源和培养-产生和不产生il - 2的细胞系(8)。在含有2 mM谷氨酰胺和5%热灭活胎牛血清的RPMI 1640培养基中固定悬浮培养。Phorbol Ester处理-细胞（1-3 × 10'）与PES孵育，通常在固定悬浮培养。孵育后，将细胞从烧瓶中倒出，并用橡皮警棍刮掉那些因接触聚乙烯而粘附在塑料上的细胞。在短时间内，细胞通过搅拌保持悬浮培养。PES是从每天新鲜配制的溶液中加入的，溶液中保存有1毫米的乙醇，温度为20℃，培养基中的最终乙醇浓度从未超过0.1%，无论添加到完整细胞中还是直接添加到反应混合物中，都不会影响蛋白激酶活性。通过台盼蓝染料排除法测定的细胞活力不受PE或乙醇浓度的影响。细胞破裂和分离- PE孵育后，细胞用RPMI 1640培养基洗涤三次。将清洗后的EL4细胞在1-2 ml匀浆缓冲液（20 m~ Tris, pH 7.5, 0.33 m蔗糖，2 m~ EDTA， 0.5 mM EGTA和2 mM PMSF）中破裂，并在配有紧密玻璃棒的Dounce匀浆器中冲泡30次。细胞质溶胶在105,000 X g下离心60分钟制备。蛋白质采用Lowry等人（34）的方法测定，以牛血清白蛋白为标准。
Methods Cell Source and Culture-IL 2-producing and-nonproducing lines of EL4 cells have been described'(8). They were maintained in stationary suspension culture in RPMI 1640 medium supplemented with 2 mM glutamine and 5% heat-inactivated fetal calf serum. Phorbol Ester Treatment-Cells (1-3 X 10') were incubated with PES, usually in stationary suspension culture. After incubation, cells were poured from the flasks and those which had adhered to the plastic as a result of PE exposure were scraped from the surface with a rubber policeman. For short time periods of treatment, cells were maintained in suspension cultured by stirring. PES were added from solutions prepared fresh daily from 1-mM stocks kept in ethanol at-20" C. Final ethanol concentrations in the medium never exceeded 0.1% and did not affect protein kinase activity when added to the intact cells or when added directly to the reaction mixture. Cell viability, as measured by trypan blue dye exclusion, was unaffected by the PE or ethanol concentrations used under the incubation conditions.Cell Rupture and Fractionation-Following incubation with PE, cells were washed three times with RPMI 1640 medium. Washed EL4 cells were ruptured in 1-2 ml of homogenization buffer (20 m~ Tris, pH 7.5, 0.33 M sucrose, 2 m~ EDTA, 0.5 mM EGTA, and 2 mM PMSF) with 30 strokes in a Dounce homogenizer fitted with a tight glass pestle. Cytosol fractions were prepared by centrifuging for 60 min at 105,000 X g. Protein was determined by the method of Lowry et al.(34), using bovine serum albumin as a standard.